This invention relates to immobilized enzymes, a process for preparing such immobilized enzymes, and the use of such immobilized enzymes to convert glucose to fructose in a continuous column process.
In conventional enzyme conversion processes, the enzymatic reaction is conducted by mixing the enzyme with the substrate, and thereafter removing the enzyme from the products or the unreacted substrate following the reaction. The difficulty in separating or recovering used enzymes has continued to be a problem. Because of the relatively high cost of enzymes, it is desirable to recover them for re-use in subsequent reactions.
Procedures for carrying out this recovery of enzyme for re-use are impractical for water soluble enzymes; recovery of whole cell enzyme is generally relatively costly and not worthwhile because of loss of significant amounts of activity in the initial isomerization.
In the conversion of glucose to fructose using glucose isomerase, because of the economics involved in producing glucose isomerase, it is of the utmost importance to use the isomerase under conditions whereby maximum yields of fructose are produced using minimum quantities of glucose isomerase. Moreover, the conditions for isomerizing should be such that minimal quantities of objectionable by-products are produced.
Various microorganisms are known in the art which produce glucose isomerase. For example, Pseudomonas hydrophila, microorganisms classified as belonging to the Streptomyces genus, such as Streptomyces flavovirens, Streptomyces achromogenes, Streptomyces echinatus and Streptomyces albus, Aerobacter cloacae, Bacillus megaterium, Acetobacter suboxydans, Acetobacter melanogenus, Acetobacter roseus, Acetobacter oxydans, Bacillus fructose and Lactobacillus fermenti are known to produce glucose isomerase.
This application is particularly concerned with use of organisms of the Actinoplanes genus, specifically Actinoplanes missouriensis, to produce glucose isomerase. This is broadly shown in Shieh et al U.S. Pat. No. 3,834,988.
Glucose isomerase is produced predominantly intracellularly by a number of the foregoing microorganisms. Thus, the major portion of the glucose isomerase is found within the cell walls of the microorganisms. Normally when these cells are used to isomerize glucose to fructose in a batch process, some loss in enzyme activity ensues and the recovery of the cells from the product for re-use of the enzyme is difficult as well as costly.
In answer to these problems, the art has developed immobilized enzymes, in which the enzymes are bound to inert or insoluble carriers and these immobilized enzymes can be used in columns to produce continuous reactions in which conversion of glucose to fructose takes place continuously in the column.
In certain cases it is advantageous to use an immobilized enzyme rather than a soluble one. The advantages of using an immobilized enzyme in comparison with one in soluble form are that the immobilized enzyme is re-usable and does not contaminate the reaction products and, therefore, is eminently suitable for continuous or repeated use. The disadvantages of the known immobilized enzymes are that some have to be prepared by rather complicated methods and, accordingly, are relatively expensive, and others require a rather large proportion of a carrier or a binder. Also some of the known immobilized enzymes suffer from the disadvantage that when packed in a column they exhibit flow properties which are not satisfactory. Moreover, a great many of the known binders are synthetic polymer products and consequently, are not suitable for production of foodstuffs.
In an article entitled "Sucrose Inversion by Immobilized Yeast Cells in a Complete Mixing Reactor" by Toda and Shoda, published in Biotechnology and Bioengineering, Vol. XVII, pages 481-497 (1975) a method of preparing a spherical agar gel is disclosed. In this process a suspension of yeast cells having invertase activity is mixed into a 2.5% (w/v) agar solution at 50.degree. C. The volume ratio of the yeast cell suspension to the agar solution is 1:4. The resulting mixture is then injected into a cold solution of toluene and tetrachloroethylene in a vertical glass tube to produce gelatinized agar pellets. The pellets were spherical and yeast cells were distributed homogeneously in the pellets.
In Zienty U.S. Pat. No. 3,779,869, a process for stabilizing glucose isomerase in whole bacterial cells is disclosed. This process comprises treating whole bacterial cells having a glucose isomerase activity at a pH from about 6.5 to about 8.5 with from about 0.1 to about 50 weight percent glutaraldehyde (based on the dry weight of the cells). However, Zienty's process results in a product suitable only for a batch process. The flow properties of the bacterial cells prevent their use in a continuous system by themselves.
In O'Driscoll et al U.S. Pat. No. 3,859,169, an enzymatically active soluble gel is formed by polymerizing to a gel a reaction mixture containing water, a water-soluble polymerizable monomer (a glycol unit), a cross-linking agent, a synthetic polymer, a free radical initiating system, and an enzyme. However, O'Driscoll did not employ glucose isomerase in his polymerized gel. Moreover, there is no mention in this patent of the use of whole cell glucose isomerase.
Moscowitz U.S. Pat. No. 3,843,442 shows immobilized glucose isomerase prepared by reacting whole microbial cells containing glucose isomerase with a diazotized aromatic primary diamino compound. This reaction is relatively complex and results in a substantial loss of activity.
Republic of South Africa application No. 73/5916 by Amotz shows immobilized glucose isomerase prepared by combining enzyme, reinforcing agent (if desired), cross-linking agent, water and/or organic solvent(s). Amotz does not disclose or claim an entrapped enzyme.
Thompson U.S. Pat. Nos. 3,788,945 and 3,909,354 disclose processes of enzymatically converting glucose to fructose by passing a glucose-containing solution through a bed of glucose isomerase which has been released from the cell and then bound to an inert carrier described in diethylaminoethyl cellulose and other porous synthetic anion-exchange resins.
Accordingly, it is the principal object of the present invention to provide a continuous process for enzymatically isomerizing glucose to fructose. A continuous system is more efficient, and consequently less expensive, than a batch system.
Another object is to provide a whole cell glucose isomerase entrapped in agar for use in a continuous process of converting glucose to fructose.
Another object is to provide a method for producing this agar entrapped whole cell glucose isomerase by means of a relatively simple process.
Still another object is to provide the aforementioned immobilized glucose isomerizing enzyme having a half life substantially longer than the half life of known non-immobilized whole cell glucose isomerizing enzymes.
A further object is to provide a method of immobilizing glucose isomerase in spherical agar particles in combination with cobaltous ions, whereby the activity of the enzyme is enhanced without the need for using cobaltous ion in the substrate.
These and other objects and advantages will become apparent hereinafter.